Microglia are critical to maintaining the internal environment of the central nervous system (CNS). These specialized resident cells function to nourish and support neurons and to act as a first line of defense in response to neuronal injury. In response to a neuropathological state, quiescent microglia undergo a series of changes that result in the release of pro-inflammatory and cytotoxic mediators for the removal of the pathogen. Upon clearance of injured cells by phagocytosis and/or the removal of toxin and toxicants, microglia return to a resting state or undergo programmed cell death. Microglia, therefore, exhibit different phenotypes depending on their surrounding environment. Expression of the appropriate phenotype is critical to the successful removal of the pathogen and to limiting damage to surrounding neurons. Chronic microglial activation has been observed in a variety of neurodegenerative diseases but, to date, it is not clear whether microglial activation is due to a persistent neuronal degeneration that warrants their activated state, or to microglial dysfunction, including a failure to either up-regulate or down-regulate the release of cytotoxic mediators including nitric oxide (NO). NO is both a potent cytotoxic mediator and a key regulator of cellular signaling within microglia. The investigator's hypothesis is that the phenotypic response of microglia to toxin exposure is dependent on the metabolic fate of NO. Redox active transition metals have been proposed as important factors in neurodegenerative diseases including Alzheimer's, Parkinson's and amiotropic lateral sclerosis. Levels of the transition metal copper are strictly regulated and deviations will alter NO signaling by changing the redox environment of the cell, particularly in reference to thiols. The investigator proposes to investigate the mechanisms by which copper alters copper- stimulated NO signaling and, thus, the phenotypic response of microglia. During the mentored phase of the award in the laboratory of Dr. Andrew Gow, the investigator will investigate the effects of copper on phenotypic differentiation in immortalized BV-2 and in primary microglia cell cultures. In particular, she will examine how copper alters key-signaling molecules and the S-nitrosylation profile in response to an acute toxin challenge and how the presence of copper might interfere with the adoption of an adaptive inflammatory phenotype. The independent phase of the award will build upon the findings obtained during the mentored phase. During this phase the investigator will investigate the effects of chronic copper overload on microglia phenotypic changes in specific anatomical brain structures in response to systemic LPS challenge in the tx j mouse. The effects of chronic copper overload will also be investigated with respect to whether the effects on microglia phenotype are permanent or can be reversed after excess copper has been removed. Public Health Relevance: Microglia are the resident immune cells in the brain where they provide the first line of defense in response to neurological insults. In response to changes in the surrounding environment, the state of activation of microglia may change, that is, they may undergo phenotypic changes. These changes are usually accompanied by the release of pro- or anti-inflammatory products that will affect the outcome of the neurological insult. In this research project the investigators propose to define the role that nitric oxide, one of the cytotoxic products of activated microglia, plays in the phenotypic differentiation of microglia and how copper (whose levels are elevated in a variety of brain pathologies), may interfere with the S-nitrosylation of protein synthesis in response to a toxin. Understanding the conditions that govern phenotypic changes in microglia may lead to the development of novel therapeutics in neurodegenerative diseases where microglia are in a chronic state of activation.